FBW7 Inhibits Myeloid Differentiation in Acute Myeloid Leukemia via GSK3-Dependent Ubiquitination of PU.1

Abstract

Glycogen synthase kinase 3β (GSK3β), an ubiquitously expressed serine/threonine kinase is reported to be overexpressed and hyperactivated in cancers including acute myeloid leukemia (AML) where it promotes self-renewal, growth, and survival of AML cells. Therefore, GSK3β inhibition results in AML cell growth inhibition and myeloid differentiation. Here we identified master transcription factor PU.1 of monocyte–macrophage differentiation pathway as potential GSK3β target. We demonstrate that GSK3β phosphorylates PU.1 at Ser41 and Ser140 leading to its recognition and subsequent ubiquitin-mediated degradation by E3 ubiquitin ligase FBW7. This GSK3-dependent degradation of PU.1 by FBW7 inhibited monocyte–macrophage differentiation. We further showed that a phospho-deficient PU.1 mutant (PU.1-S41, S140A) neither bound to FBW7 nor was degraded by it. Consequently, PU.1-S41, S140A retained its transactivation, DNA-binding ability and promoted monocyte–macrophage differentiation of U937 cells even without phorbol 12-myristate 13-acetate (PMA) treatment. We further showed that FBW7 overexpression inhibited both PMA as well as M-CSF–induced macrophage differentiation of myeloid cell lines and peripheral blood mononuclear cells (PBMC) from healthy volunteers, respectively. Contrarily, FBW7 depletion promoted differentiation of these cells even without any inducer suggesting for a robust role of GSK3β–FBW7 axis in negatively regulating myeloid differentiation. Furthermore, we also recapitulated these findings in PBMCs isolated from patients with leukemia where FBW7 overexpression markedly inhibited endogenous PU.1 protein levels. In addition, PBMCs also showed enhanced differentiation when treated with M-CSF and GSK3 inhibitor (SB216763) together compared with M-CSF treatment alone. Implications: Our data demonstrate a plausible mechanism behind PU.1 restoration and induction of myeloid differentiation upon GSK3β inhibition and further substantiates potential of GSK3β as a therapeutic target in AML.

Ubiquitin-Mediated Control of ETS Transcription Factors: Roles in Cancer and Development

“…PU.1 is notable because within the haematopoietic lineage it drives differentiation of precursor cells into monocytes and macrophages [ 62 ]. In this instance, inhibition of FBXW7 or enhancement of USP22 activity towards PU.1 promoted precursor differentiation [ 63 , 65 ]. The other exception is the SAM pointed domain-containing ETS transcription factor (SPDEF), the only member of the PDEF subfamily, whose anti-metastatic and anti-tumorigenic activities in PCa and hepatocellular carcinoma cells respectively are antagonised by UPS processing following phosphorylation by CDK11B [ 107 , 108 ].…”

“…PU.1 (or SPI-1, of the SPI subfamily) plays an essential role in lineage commitment of haematopoietic precursor cells into macrophages and monocytes whereby impaired PU.1 function has been linked to the development of acute myeloid leukaemia (AML) [ 62 ]. In common with ERG, PU.1 contains two phosphodegrons that can be phosphorylated by GSK-3β and subsequently bound by FBXW7, leading to its ubiquitination and degradation ( Figure 1 a) [ 63 ]. Blockade of the GSK-3β-FBXW7 signalling axis restored PU.1 levels in peripheral blood mononuclear cells and promoted their differentiation.…”

Transcription Factors, R-Loops and Deubiquitinating Enzymes: Emerging Targets in Myelodysplastic Syndromes and Acute Myeloid Leukemia

“…This suggests that a modest PU.1 reduction can act as driver mutation [124]. Interestingly, the reduction in PU1 and CEBPa levels caused by aberrant ubiquitination signals have been recently described in AML, further demonstrating the importance of a proper TF protein dosage [125,126].…”

Acute Myeloid Leukemia-Related Proteins Modified by Ubiquitin and Ubiquitin-like Proteins

“…Furthermore, the GSK3β phosphorylation consensus motif (S/TXXXS/T) often coincides with the FBWX7 phosphodegron motifs [ 68 ]. GSK3β phosphorylates PU.1 at serine41 and serine140, leading to its recognition and subsequent ubiquitin-mediated degradation by Ub E3 ligase FBXW7 [ 69 ].…”